Inhibition of microbial biofuel production in drought-stressed switchgrass hydrolysate

Abstract

Background: Interannual variability in precipitation, particularly drought, can affect lignocellulosic crop biomass yields and composition, and is expected to increase biofuel yield variability. However, the effect of precipitation on downstream fermentation processes has never been directly characterized. In order to investigate the impact of interannual climate variability on biofuel production, corn stover and switchgrass were collected during 3 years with significantly different precipitation profiles, representing a major drought year (2012) and 2 years with average precipitation for the entire season (2010 and 2013). All feedstocks were AFEX (ammonia fiber expansion)-pretreated, enzymatically hydrolyzed, and the hydrolysates separately fermented using xylose-utilizing strains of Saccharomyces cerevisiae and Zymomonas mobilis. A chemical genomics approach was also used to evaluate the growth of yeast mutants in the hydrolysates.

Results: While most corn stover and switchgrass hydrolysates were readily fermented, growth of S. cerevisiae was completely inhibited in hydrolysate generated from drought-stressed switchgrass. Based on chemical genomics analysis, yeast strains deficient in genes related to protein trafficking within the cell were significantly more resistant to the drought-year switchgrass hydrolysate. Detailed biomass and hydrolysate characterization revealed that switchgrass accumulated greater concentrations of soluble sugars in response to the drought and these sugars were subsequently degraded to pyrazines and imidazoles during ammonia-based pretreatment. When added ex situ to normal switchgrass hydrolysate, imidazoles and pyrazines caused anaerobic growth inhibition of S. cerevisiae.

Conclusions: In response to the osmotic pressures experienced during drought stress, plants accumulate soluble sugars that are susceptible to degradation during chemical pretreatments. For ammonia-based pretreatment, these sugars degrade to imidazoles and pyrazines. These compounds contribute to S. cerevisiae growth inhibition in drought-year switchgrass hydrolysate. This work discovered that variation in environmental conditions during the growth of bioenergy crops could have significant detrimental effects on fermentation organisms during biofuel production. These findings are relevant to regions where climate change is predicted to cause an increased incidence of drought and to marginal lands with poor water-holding capacity, where fluctuations in soil moisture may trigger frequent drought stress response in lignocellulosic feedstocks.

Keywords: Biofuel; Corn stover; Drought; Fermentation inhibition; Lignocellulose; Saccharomyces cerevisiae; Switchgrass.

Fig. 1

Interannual weather variation. a Temperature [growing degree days (GDD)] and b precipitation for 2010, 2012, and 2013, and the 30-year average values at Arlington Research Station in south central Wisconsin (ARL, 43˚17′45″ N, 89˚22′48″ W, 315 masl)

Fig. 2

Fermentation profiles for Zymomonas mobilis 2032 grown in corn stover and switchgrass hydrolysates from different harvest years. a 2010 ACSH (36H56), b 2012 ACSH (36H56), c 2012 ACSH (P0448R), d 2013 ACSH (P0448R), e 2010 ASGH (Shawnee), f 2012 ASGH (Shawnee), g 2013 ASGH [Cave-in-Rock (CIR)]. Data points represent the mean ± SD (n = 3). Error bars that are smaller than the individual data points may be hidden from view

Fig. 3

Fermentation profiles for Saccharomyces cerevisiae Y128 grown in AFEX-treated biomass hydrolysates. a 2010 ACSH (36H56), b 2012 ACSH (36H56), c 2012 ACSH (P0448R), d 2013 ACSH (P0448R), e 2010 ASGH (Shawnee), f 2012 ASGH (Shawnee), g 2013 ASGH [Cave-in-Rock (CIR)]. Data points represent the mean ± SD (n = 3). Error bars that are smaller than the individual data points may be hidden from view

Fig. 4

Chemical genomic analysis of hydrolysate variation. a Fitness heat map for yeast mutants in corn stover (CS) and switchgrass (SG) hydrolysates. The genome-wide yeast deletion mutant collection was grown in fifteen different hydrolysate batches (n = 3 per feedstock) and a synthetic hydrolysate (SynH2.1) control (n = 3). The abundance of each mutant was assessed by sequencing the strain-specific barcodes and a fitness score was determined relative to the synthetic hydrolysate control. Mutants sensitive to the hydrolysate conditions are shown in blue and resistant are shown in yellow, compared to the abundance in the SynH2.1 control. The (3) represents the 36H56 variety and the (P) represents the P0448R variety of corn stover. b Fitness plot of yeast mutants grown in 2012 ASGH. The most resistant (fitness > 4) and susceptible mutants (fitness < −5) are labeled and shown in red. c Intersection of yeast mutants that are highly susceptible or resistant to all hydrolysates, only the 2012 ASGH, or all hydrolysates except the 2012 ASGH. *The fitness of these mutants was statistically different (p < 0.001) in the 2012 ASGH versus the other four hydrolysates [2013 ASGH, 2010 CS (36H56), 2012 CS (P0448R), 2013 CS (P0448R)]

Fig. 5

Untreated biomass composition. a Water and ethanol soluble extractives. b Structural carbohydrates and lignin. Values are reported as the mean ± SD (n = 3)

Fig. 6

Principal component analysis (PCA) of hydrolysate composition data—relationship between principal components 1 and 2. a Hydrolysate batches grouped by plant variety. b Hydrolysate batches grouped by year. c Correlation score graph showing relative effect of each hydrolysate component

Fig. 7

Imidazole and pyrazine detection and quantification in AFEX-treated biomass and hydrolysates. a Overlaid mass spectrometric chromatogram of ethyl acetate extracts of AFEX-treated switchgrass hydrolysates. Each line represents a replicate batch of hydrolysate (2012: n = 3; 2010 and 2013: n = 2). b Imidazole and pyrazine content of untreated and AFEX-treated corn stover (CS) and switchgrass (SG). c Correlation between imidazole and pyrazine content of AFEX-treated biomass and untreated biomass soluble sugars (sucrose, glucose fructose, xylose, arabinose, and galactose)

Fig. 8

Imidazoles and pyrazines found in drought-year AFEX-treated switchgrass hydrolysate (ASGH) can impair anaerobic yeast growth. Anaerobic yeast growth in add-back experiment, with various concentrations of pyrazines and imidazoles (P/I) in 2010 ASGH relative to estimated levels in 2012 ASGH (mean, n = 3). Average cell densities with standard error of the mean are reported from triplicate samples, with every twelfth time point plotted (roughly one time point every 2 h)

Fig. 9

Interaction between plant response to environmental conditions and pretreatment chemistry. In lignocellulosic biomass, drought stress causes an increase in osmoprotectants, including soluble sugars that are degraded to microbial inhibitors during thermochemical pretreatments